The level of expression of a protein can be affected by several different mechanisms following transcription initiation. For example, the level of expression of the Int gene of bacteriophage P2 has been shown to be controlled by three different mechanisms (Yu et al., J. Virol. 1994, 68(7), 4220-6). First, a partial transcription termination signal located between the Int and C genes reduces the transcriptional readthrough by about 30%. Second, the ribosome binding site and AUG codon of the Int gene are located in a putative stem loop structure which may inhibit the initiation of translation. The third control of int expression in P2 seems to be posttranscription autoregulation wherein the binding site of the int protein on Int gene mRNA is shown to extend into the ribosome binding site of Int, supporting a competitive binding theory between Int and ribosomes. It has been recognized, however, that translation is a very important control step in gene expression.
It is believed that at least one of the functions of the secondary and tertiary structure of RNA is the control of translation. Mutations which prevent the formation of secondary structures such as stem loops have been shown to increase expression of a protein up to 20 fold (Wikstrom et al., J. Mol Biol. 1992 224(4), 949-966). In addition, insertion of stem loop structures into a gene expression system such as Saccharomyces cerevisiae has been shown to inhibit translation up to 89% (Oliveira et al. Mol. Microbiol. 1993 9(3), 521-532).
One mechanism by which translational control is thought to occur is by regulation of ribosome movement down the mRNA by the specific binding of cytosolic proteins to the RNA structure. It is believed that for many proteins, this binding occurs in the secondary structure such as a stem loop structure. For example, intracellular iron can be stored in the protein shell of ferritin to protect the cell against the toxic action of the iron. In response to increased iron, some ferritin subunits are synthesized using translation and transcriptional mechanisms. Translational control has been shown to involve a unique stem loop structure in the 5' untranslated region of the subunit messengers (Bomford et al. Pathobiology 1992, 60(1), 10-18). When the iron level is low, a protein binds to this stem loop structure and prevents translation. When intracellular iron levels rise, the repressor protein is discharged and the large population of messengers begins to translate.
It has also been shown that the Epstein-Barr virus small RNA species, EBER-1, controls protein synthesis in a similar fashion (Clark et al. Eur. J. Biochem. 1990, 193(3), 635-641). It was found that EBER-1 prevents inhibition of protein synthesis caused by low concentrations of synthetic double-stranded RNA. This effect was eliminated by disruption of the secondary structure of EBER-1. Thus, it was suggested that the ability of EBER-1 to regulate protein synthesis is dependent upon the secondary structure of the RNA molecule.
The transient disruption of the secondary RNA structure has also been implicated as a primary step in the expression of proteins due to heat shock (Nagai et al. Proc. Natl. Acad. Sci. USA 1991, 88(23), 10515-9).
It has now been found that administration of at least a portion of a stem loop structure of an mRNA to a cell or tissue can modulate the synthesis of a selected protein in the cell or tissue.